In order to establish measures against environmental pollution that is one of serious problems of modern society, it is necessary to accurately assess a degree of exposure to a hazardous chemical substance in the environment. It is thus indispensable that a method has been established of analyzing a hazardous chemical substance with ease, high sensitivity and high reproducibility.
Even a minute level of dioxins causes various kinds of hazardous action. Accordingly, the establishment of the method of sensing dioxins sharply and quickly is thought to be an urgent issue to assess exposure to dioxins accurately and prevent health problems. Further, it is a significant issue to totally and quantitatively assess biological toxicity of polycyclic aromatic hydrocarbon that is a hazardous substance in the environment as well as dioxins, and tobacco smoke including polycyclic aromatic hydrocarbon and dioxins as a complex.
Typical methods of sensing dioxins and the like in the environment include a method using an endogenous biomarker such as, for example, cytochrome P-4501A1 that is a drug metabolizing enzyme, bioassay using cultured cells, enzyme immunoassay, method using chromatography, and the like. Particularly, the bioassay using gene engineering techniques has recently attracted attention due to the convenience and high sensitivity.
Such a gene-engineering bioassay is comprised of some basic units. The basic units are two i.e. (1) cell and (2) gene structure integrated into the cell. The gene structure is comprised of a dioxins responsive DNA sequence that is a DNA sequence functioning as a sensor for dioxins, and a marker protein gene that specifies a marker protein.
By introducing the gene structure to a cell to stably integrate into chromosome DNA of the cell, it is possible to generate a sensor cell reacting with dioxins and the like.
When such a transgenic cell is exposed to a sample including dioxins or the like, the dioxins or the like first react with AhR in the cell, and further, form a complex with a coactivator (hereinafter abbreviated as Arnt) that is a transcription promoting coupling factor, and the complex activates the dioxins responsive DNA sequence as a transcription factor. As a result, expression of the marker protein gene is accelerated downstream of the gene sequence.
By expression of the marker protein gene, the maker protein is produced, and by quantitatively determining the protein, it is possible to assess that what concentration of dioxins or the like exists in the sample.
As the marker protein gene, used conventionally are enzyme genes such as chloramphenicol acetyl transferase, β-galactosidase, luciferase and the like, and green fluorescent protein genes.
However, the conventional bioassay has problems that (1) since a marker protein is not secreted, the operation is required of cutting the cell to extract the marker protein for determination of the protein, (2) the sensitivity is not sufficient in sensing a minute level of a hazardous substance in the environment, and even in the high-sensitive system, 1 pM is a sensing limit in terms of 2,3,7,8-tetrachlorodibenzo-p-dioxin (hereinafter, abbreviated as TCDD), (3) two or three days are required for a bioassay, (4) 60,000 to 100,000 cells are required for one sample, and therefore, costs such as cultivation costs and personnel costs are high. Particularly, items (2) to (4) are serious problems in efficiently screening a large number of samples.
Non-patent Document 1: P. A. Behnisch, K. Hosoe, S. Sakai, Bioanalytical screening methods for dioxins and dioxin-like compounds: a review of bioassay/biomarker technology, Environ. Int. 27 (2001) 413-439.
Non-patent Document 2: Japanese Patent Application No. 2004-135662 “Dioxins measurement transformant and sensing method, quantitative analysis method and screening method for dioxins using the transformant”